It is generally accepted that for an individual species to co-exist indefinitely with the thousands of species that make up the intestinal microbiota it must use one or a few limiting carbon sources better than all other species (nutrient-niche hypothesis). The nutrient niche hypothesis predicts competition for resources from a nutrient mixture, but has never been adequately tested. Thus, we will thoroughly test the nutrient-niche hypothesis for Escherichia coli in the streptomycin-treated mouse model, which is the only animal model of intestinal colonization that allows competition between experimentally introduced bacteria and a rich resident microbial community. This proposal builds on three important discoveries made in our laboratories: a) that different E. coli strains differ in their in vivo sugar preferences, despite being alike in their in vitro preferences for the same sugars b) that several human commensal E. coli strains overcome colonization resistance to grow from low to high numbers and co-colonize mice that are pre-colonized with another E. coli strain, and c) that a combination of different human commensal E. coli strains can completely prevent E. coli EDL933, an O157:H7 strain, from colonizing. We will determine whether different E. coli strains compete directly with one another and the microbiota for specific carbon sources from a mixture of nutrients that is equally available to all members of the microbiota, as the nutrient-niche hypothesis assumes, by virtue of having a competitive advantage resulting from allelic differences between their sugar catabolism regulons. The proposed studies will distinguish whether competition for resources between closely related E. coli strains and the microbiota is direct or indirect;the former indicating simple competition for sugars from a nutrient mixture provided by the host and cooperative metabolism of the normal microbiota, the latter indicating more complex interactions of the different E. coli strains with various members of the microbiota and the host, e.g., interactions that produce a unique set of carbon sources for consumption by that specific E. coli strain. In Aim 1, we will use operon swapping between E. coli strains to identify gene systems conferring colonization advantages that allow individual E. coli strains to execute different nutritional programs to compete with the normal microbiota in the intestine. Aim 2 is to determine how different strains of E. coli compete with one another for nutrients in the intestine, using the same genomics-based approach to identify the gene systems that confer colonization advantages. In Aim 3, we will determine if other E. coli pathogens use the same nutritional program as E. coli EDL933 in the intestine. These aims focus on competition for carbon and energy sources in the intestine, which is fundamental to colonization by the entire microbiota. The overall impact of this application is that it will test the nutrient-niche hypothesis to advance our understanding of how the normal microbiota is established, maintained, and forms a barrier to disease. This knowledge is crucial for guiding rational design of E. coli probiotic strains.